The main objective of this work is to elucidate the biochemical mechanisms regulating the GTP-binding protein ARF and, in so doing, to further our understanding of the role of ARF in membrane traffic. We found that the amino terminal alpha helix of ARF and covalently bound myristate form a GTP- and phospholipid-sensitive switch and that this domain is necessary for interaction with ARF GAP and Gs. We are currently testing the hypothesis that this is an effector domain of the protein. ARF's interaction with target proteins is dependent on phospholipids. We found that ARF has both nonspecific interactions with lipids and detergents and a specific interaction with phosphatidylinositol 4,5- bisphosphate (PIP2). These phospholipid interactions could be dissociated. The amino terminus of ARF was necessary for the nonspecific association with phospholipids but was not involved in specific interactions with PIP2. Using mutational analysis we have preliminary data implicating several specific residues of ARF in PIP2 binding. We are further testing the hypothesis that ARF is a PIP2 binding protein and the related hypothesis that PIP2 acts as a cofactor for ARF interaction with target proteins. Having found that PIP2 and PA were required for ARF - ARF GAP interaction, we have been able to make some progress in the purification of the latter protein. ARF GTPase activating protein copurified with clathrin assembly protein complex 2. We are currently testing the hypothesis that AP-2 is ARF GAP. These data support the hypothesis that the binding and hydrolysis of GTP by ARF regulates the assembly of coat proteins on transport vesicles.